Abstract Glucocorticoids (GC) play a key role in metabolic adaptation during stress. However, chronic exposure to GC, is harmful and results in disorders such as insulin resistance. We previously identified angiopoietin-like 4 (Angptl4) as a GC-regulated gene encoding a secreted protein that promotes adipocyte lipolysis and inhibits extracellular lipoprotein lipase (LPL). We showed that Angptl4 is required for GC-promoted lipolysis in white adipose tissue (WAT) and in Angptl4 null mice (Angptl4-/-), excess GC-induced fatty liver and hypertriglyceridemia are diminished. Our recent preliminary studies further demonstrated that GC-induced insulin resistance was compromised in Angptl4-/- mice. Intriguingly, GC treatment increased hepatic ceramide levels, but such GC effect was attenuated in the absence of Angptl4. Indeed, myriocin, which inhibits ceramide synthesis, decreased GC-induced insulin resistance in wild type (WT) but not Angptl4-/- mice. We additionally found that GC exposure elevated the activity of PP2A and PKC? (two downstream effectors of ceramides) as well as the expression of a list of ceramide synthetic genes in liver. In Angptl4-/- mice, the activity of PP2A and PKC? was mitigated and the expression of ceramide synthase 3-6 (Cers3-6) was specifically impaired. Based on these preliminary data, we hypothesize that GC augment the expression of Angptl4, which promotes WAT lipolysis mobilizing fatty acids (FA) to liver to serve as substrates and signals required for GC to increase Cers3-6 expression. This elevates the production of specific ceramide species to activate PP2A and PKC? to suppress insulin action. In this proposal, we will examine this model. In Aim 1, our preliminary study found that the fibrinogen-like domain (FLD) of Angptl4 promotes adipocyte lipolysis without inhibiting LPL. We will test whether FLD restores GC-induced insulin resistance in Angptl4-/- mice. We will also examine the role of FA mobilization to the liver in GC-induced insulin resistance using mice with reduced expression of two major hepatic FA transporters, Fatp5 and Cd36. Moreover, hyperinsulinemic- euglycemic clamps will be used to determine the role of Angptl4 in GC-augmented hepatic glucose production, a hallmark of hepatic insulin resistance. In Aim 2, we will determine which ceramide synthase(s), Cers3-6, are involved in GC-induced insulin resistance. PPAR? can be activated by FA and has been shown to increase ceramide synthesis in certain tissues. Thus, its role in GC-induced hepatic Cers3-6 expression and ceramide production will be examined. In Aim 3, we will analyze the role of PP2A and PKC? in GC- induced insulin resistance and will identify ceramide synthase(s) that activate PP2A and PKC? to cause insulin resistance. Overall, this study will be the first to establish the FLD of Angptl4 as a lipid mobilization factor participating in GC-induced insulin resistance and for the first time, specific ceramide synthases that activate PKC? and PP2A will be identified. These findings will advance our understanding of the mechanism of insulin resistance and will provide novel molecular targets for improving insulin sensitivity. ! !